U.S. patent number 4,769,181 [Application Number 06/549,047] was granted by the patent office on 1988-09-06 for 1,25-dihydroxyvitamin d.sub.2 compounds.
This patent grant is currently assigned to Wisconsin Alumni Research Foundation. Invention is credited to Hector F. DeLuca, Heinrich K. Schnoes, Rafal R. Sicinski, Yoko Tanaka.
United States Patent |
4,769,181 |
DeLuca , et al. |
September 6, 1988 |
**Please see images for:
( Certificate of Correction ) ** |
1,25-dihydroxyvitamin D.sub.2 compounds
Abstract
The invention is directed to the preparation of hydroxylated
compounds of the vitamin D.sub.2 series and specifically to a
process for synthesizing 1.alpha.,25-dihydroxyvitamin D.sub.2,
1.beta.,25-dihydroxyvitamin D.sub.2, their corresponding 5,6-trans
isomers and the C-24 epimers of these compounds. The hydroxylated
vitamin D.sub.2 compounds obtained exhibit vitamin D-like activity
and can be substituted for vitamin D.sub.3 or various of its known
metabolites where such compounds are applied.
Inventors: |
DeLuca; Hector F. (Madison,
WI), Schnoes; Heinrich K. (Madison, WI), Sicinski; Rafal
R. (Madison, WI), Tanaka; Yoko (Madison, WI) |
Assignee: |
Wisconsin Alumni Research
Foundation (Madison, WI)
|
Family
ID: |
24191451 |
Appl.
No.: |
06/549,047 |
Filed: |
November 7, 1983 |
Current U.S.
Class: |
552/653; 540/116;
552/501; 540/51; 549/453 |
Current CPC
Class: |
C07J
71/0042 (20130101); C07C 401/00 (20130101) |
Current International
Class: |
C07C
401/00 (20060101); C07J 71/00 (20060101); C07J
009/00 () |
Field of
Search: |
;260/397.2 ;424/236 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Schenkman; Leonard
Assistant Examiner: Lipovsky; Joseph A.
Attorney, Agent or Firm: Bremer; Howard W.
Government Interests
This invention was made with Government support under NIH Grant No.
AM 14881 awarded by the Department of Health and Human Services.
The Government has certain rights in this invention.
Claims
We claim:
1. A compound having the formula:
where the methyl group at carbon 24 may have the R or S
configuration.
Description
TECHNICAL FIELD
This invention relates to the preparation of hydroxylated compounds
of the vitamin D.sub.2 series.
More specifically, this invention relates to a process for the
synthesis of 1.alpha.,25-dihydroxyvitamin D.sub.2 and
1.beta.,25-dihydroxyvitamin D.sub.2, of the corresponding
5,6-trans-isomers, and the C-24 epimers of these compounds.
BACKGROUND ART
The importance of hydroxylated forms of vitamin D as regulators of
calcium and phosphate metabolism in animals and humans is by now
well established through many disclosures in the patent and general
literature. Vitamin D.sub.3, the natural form of the vitamin
produced in skin, is known to be hydroxylated in vivo to
25-hydroxyvitamin D.sub.3 and then to 1.alpha.,25-dihydroxyvitamin
D.sub.3, the latter compound being generally regarded as the
tissue-active hormonal form of the vitamin. Likewise, vitamin
D.sub.2, which is commonly used as a food additive or vitamin D
supplement, undergoes the same hydroxylation sequence in vivo to
form 25-hydroxyvitamin D.sub.2 (25-OH-D.sub.2) and
1.alpha.,25-dihydroxyvitamin D.sub.2 (1.alpha.,25-(OH).sub.2
D.sub.2), the latter being essentially as active as
1.alpha.,25-dihydroxyvitamin D.sub.3 in humans and other
mammals.
Both 25-OH-D.sub.2 and 1.alpha.,25-(OH).sub.2 D.sub.2, the
structures of which are shown below, have been isolated and
characterized as metabolites of vitamin D.sub.2 (DeLuca et al.,
U.S. Pat Nos. 3,585,221; 3,880,894). ##STR1## These metabolites,
being derived from vitamin D.sub.2, are characterized by the
S-stereochemistry at carbon 24, as shown above.
DISCLOSURE OF INVENTION
A process for the preparation of 1,25-dihydroxyvitamin D.sub.2
compounds has now been developed. The process is notable in that,
in addition to the 5,6-cis- and trans-1.alpha.,25-dihydroxy vitamin
D.sub.2 compounds, it also provides 1.beta.,25-dihydroxyvitamin
D.sub.2 compounds and the corresponding 5,6-trans isomers. These
latter analogs are of particular interest because of their
unexpectedly potent biological properties.
The synthetic process is outlined in Scheme I. Specific compound
designations by Arabic numeral or letter (e.g. 1, 2, . . . 6a, 6b,
etc.) as used in the following description or the Examples refer to
the structures so numbered in Scheme I or in the body of this
disclosure. A wavy line drawn to a substituent in these structures,
e.g. as in the case of the substituent at carbons 1 and 24,
indicates that such substituent may have either of the two possible
stereochemical configurations.
BEST MODE FOR CARRYING OUT THE INVENTION
A convenient starting material for this process is
25-hydroxyvitamin D.sub.2 (structure 1, 24S-sterochemistry), or its
24R-epimer, 25-hydroxy-24-epi-vitamin D.sub.2 (structure 1,
24R-stereochemistry), or a mixture of both compounds. ##STR2## Both
25-hydroxyvitamin D.sub.2, having the natural 24S-configuration,
and its 24R-epimer ia a known compound. The 24R-epimer has been
prepared previously (DeLuca, U.S. Pat. No. 3,585,221 and DeLuca et
al. U.S. patent application Ser. No. 420,191, filed Sept. 20,
1982). For the purpose of the present process it is convenient to
use a mixture of the 24R and S-epimers as starting material, epimer
separation being accomplished at a later stage (as described
below). It is important to note, however, that the synthetic
process can be executed equally well and in an entirely analogous
fashion with the pure 24R or 23S-epimer of compound 1 as starting
material, whereby, of course, the use of the 24R-epimer would yield
specifically the 24R-epimer of the 1,25-dihydroxyvitamin D.sub.2
product, whereas the use of the 24S-epimer would yield the
corresponding 24S-1,25-dihydroxyvitamin D.sub.2 product.
The first step of the process comprises conversion of starting
material of structure 1 to the corresponding C-3-tosylate
(structure 2) using conventional tosylation procedures, and
subsequent solvolysis of the tosylate according to the procedures
of DeLuca et al., (U.S. Pat. No. 4,195,027) to obtain the
cyclovitamin D derivative of structure 3. This intermediate is then
subjected to allylic hydroxylation with SeO.sub.2 /t-butyl
hydroperoxide, to obtain the 1-hydroxy-cyclovitamin D.sub.2
derivatives. This allylic hydroxylation process yields the
1.alpha.-hydroxy-cyclovitamin D product represented by structure 4
as expected, but also, in this case, the corresponding
1.beta.-hydroxy isomer of structure 5. Formation of the
1.beta.-hydroxy-isomer in this reaction was unexpected, since in
previous work (U.S. Pat. No. 4,195,027) only the 1.alpha.-hydroxy
product had been recovered. The 1.beta.-hydroxy compound 5 is the
minor component of the product mixture. If desired, isomer
separation can be accomplished by conventional methods, e.g. high
performance chromatography, but for the purposes of the present
process, isomer separation is not required at this stage.
Direct solvolysis of this 1-hydroxy-cyclovitamin D.sub.2 product
mixture (compounds 4 and 5) in a medium containing a low-molecular
weight organic acid, then provides, in admixture, the desired
5,6-cis- and 5,6-trans-1,25-dihydroxyvitamin D.sub.2 compounds as
the 3-O-acylates, namely the compounds of structures 6a/b and the
corresponding 5,6-trans-isomers (structure 8a/b) and the
1.beta.-hydroxy-epimers of structures 7a/b as well as their
corresponding 5,6-trans-isomers (9a/b). After initial fractionation
of this mixture by conventional high performance liquid
chromatography, followed by mild base hydrolysis of each fraction
(to remove the acyl groups) and subsequent further separation and
purification of products by high performance chromatography, it is
possible to obtain any or all of the following compounds in
isolated form: 1.alpha.,25-dihydroxyvitamin D.sub.2 (10a), the
natural product having the 24S-stereochemistry), the corresponding
(24R)-epimer, 1.alpha.,25-dihydroxy-24-epivitamin D.sub.2 (10b),
5,6-trans-1.alpha.,25-dihydroxyvitamin D.sub.2 (12a), the
corresponding (24R)-epimer ( 12b), as well as
1.beta.,25-dihydroxyvitamin D.sub.2 (11a; 24S-stereochemistry) and
its (24R)-epimer, 1.beta.,25-dihydroxy-24-epi-vitamin D.sub.2
(compound 11b), and the corresponding 5,6-trans-isomers 13a and
13b.
In the present case, compounds 10a, 10b, 11a, 11b, 12a and 12b were
recovered by chromatographic separation of the reaction mixture.
The 5,6-trans compounds 13a and 13b can also be isolated from the
solvolysis mixture by careful chromatography, but when their
abundance in the mixture is low so as to make recovery tedious and
time-consuming, it is generally more convenient to obtain them from
the cis-isomers 11a and 11b by 5,6-double bond isomerization.
Thus, treatment of the 5,6-cis isomer 11a with iodine, according to
the conventional procedure, gave upon chromatographic purification,
the 5,6-trans isomer 13a; compound 11b was likewise converted to
its trans isomer 13b.
In order to securely establish the C-24 stereochemistry of the
novel 1.beta.,25-dihydroxyvitamin D.sub.2 compounds, these
1.beta.-isomers were also chemically correlated with the known
1.alpha.-hydroxy compounds by the following procedure. Treatment of
1.alpha.,25-dihydroxyvitamin D.sub.2 (10a) with manganese dioxide,
gave the corresponding 1-oxo-25-hydroxyprevitamin D.sub.2 compound
of structure 14a, shown below: ##STR3## Upon reduction of this
compound with LiAlH.sub.4, and subsequent thermal isomerization of
the previtamin chromophore to the vitamin D triene system, there
was obtained 1.beta.,25-dihydroxy-vitamin D.sub.2 (structure 11a).
Like treatment of 1.alpha.,25-dihydroxy-24-epivitamin D.sub.2
(structure 10b) gave ketone 14b (above) and then
1.beta.,25-dihydroxy-24-epivitamin D.sub.2 (structure 11b). These
interconversions, and the cis to trans conversions (11a,b to 13a,b)
described earlier, relate all four 1.beta.,25-dihydroxyvitamin D
compounds with the corresponding 1.alpha.-hydroxy-isomers and thus
establish the C-24 stereochemistry for all compounds.
Although for therapeutic applications, the free hydroxy compounds
represented by structures 10, 11, 12 and 13 are generally used, for
some such applications, the corresponding hydroxy-protected
derivatives may be useful or preferred. Such hydroxy-protected
derivatives are for example the acylated compounds represented by
general formulae 15 and 16 below, ##STR4## wherein each of R.sub.1,
R.sub.2, and R.sub.3 is selected from the group onsisting of
hydrogen and acyl, except that at least one of R.sub.1, R.sub.2 and
R.sub.3 must be acyl. The term `acyl` refers to an aliphatic acyl
group (alkanoyl group) of from 1 to 6 carbons, in all possible
isomeric forms (e.g. formyl, acetyl, butyryl, isobutyryl, valeryl,
etc.) or an aromatic acyl group (aroyl group), such as benzoyl, or
the methyl- halo- or nitro-substituted benzoyl groups, or a
dicarboxylic acyl group of from 2 to 6 atoms chain length, i.e.
acyl groups of the type ROOC(CH.sub.2).sub.n CO--, or ROOCCH.sub.2
--O--CH.sub.2 CO--, where n has values between 0 and 4 inclusive,
and R is hydrogen or an alkyl radical. Representative of such
dicarboxylic acyl groups are oxalyl, malonyl, succinoyl, glutaryl,
adipyl and diglycolyl. The term `alkyl` refers to a lower alkyl
group of 1 to 6 carbons in all possible isomeric forms, e.g.
methyl, ethyl, propyl, isopropyl, isobutyl, pentyl, etc.
Such acyl derivatives are conveniently prepared from the free
hydroxy compounds (or, if desired, from the C-3-monoacylated
intermediates of structure 6, 7, 8 or 9) by conventional acylation
procedures, i.e. treatment of any of the hydroxyvitamin D.sub.2
products with an acyl chloride, or acyl anhydride in a suitable
solvent such as pyridine, or alkylpyridine. By appropriate
selection of reaction time, acylating agent, temperature and
solvent, as is well-known in the art, the partially or fully
acylated derivatives represented by structures 15 or 16 above are
obtained. For example, treatment of 1.beta.,25-dihydroxyvitamin
D.sub.2 in pyridine solvent with acetic anhydride at room
temperature gives the 1,3-diacetate (15, R.sub.1 =R.sub.2 =acetyl,
R.sub.3 =H), while the same reaction conducted at elevated
temperature yields the corresponding 1,3,25-triacetate. The
1,3-diacetate can be further acylated at C-25 with a different acyl
group; e.g. treatment with benzoyl chloride or succinic anhydride
would provide the 1,3-diacetyl-25-benzoyl- or
.beta.,3-diacetyl-25-succinoyl-derivative, respectively. A
1,3,25-triacyl derivative can be selectively hydrolyzed in mild
base to provide the 1,3-dihydroxy-25-acyl compound, the free
hydroxy groups of which can be reacylated, if desired, with
different acyl groups. Likewise, a 1,3-diacyl derivative can be
subjected to partial acyl hydrolysis to obtain the 1-O-acyl and the
3-O-acyl-compounds, which in turn can be reacylated with different
acyl groups. Like treatment of the other hydroxyvitamin D.sub.2
products (10, 11, 12 or 13) provides the corresponding desired acyl
derivatives of structures 15 or 16.
Like the previously known vitamin D.sub.2 metabolite,
1.alpha.,25-dihydroxyvitamin D.sub.2 (10a), the novel compounds of
this invention exhibit pronounced vitamin D-like activity, and thus
represent desirable substitutes for the known vitamin D.sub.2 or
D.sub.3 metabolites in many therapeutic or veterinary applications.
Particularly preferred in this regard are the products of structure
13a and 13b. These compounds exhibit high binding affinity for the
intestinal receptor protein, and since binding affinity generally
correlates with high in vivo activity, these compounds can be
expected to be especially useful for the treatment of diseases
related to mineral imbalance. The novel compounds may be used for
correcting or improving a variety of calcium and phosphate
imbalance conditions resulting from a variety of diseases, such as
vitamin D-resistant rickets, osteomalacia, hypoparathyroidism,
pseudohypoparathyroidism, osteoporosis, Paget's disease, and
similar bone and mineral-related disease states well known to the
medical practice. The compounds can also be used for the treatment
of mineral imbalance conditions in animals, such as the milk fever
condition, poultry or swine leg weakness, or for improving egg
shell quality of fowl.
For therapeutic purposes, these compounds may be administered
orally or by injection or infusion in any form convenient or
appropriate to the method of administration selected. Thus the
compounds may be formulated with any therapeutically acceptable and
innocuous carrier, in the form of pills, tablets or gelatin
capsules for oral administration, or they may be formulated as
solutions, emulsions, dispersions or suspensions in innocuous
solvents and oils, and such formulations may contain also other
therapeutically active and beneficial constituents, such as other
vitamins, salts, sugars, hormones, etc. as may be appropriate to
the specific application. The novel compounds may be administered
singly or as mixtures, e.g. mixtures of 13a and 13b, or mixtures of
11a, 11b, 13a, 13b, or any other combination of the individual
stereoisomers of this invention. Advantageously, the compounds of
this invention are administered in dosage amounts of between about
0.5 to 100 .mu.m per day, it being understood, of course, that the
specific dosage administered in any given case will be adjusted in
accordance with the specific compound administered, the disease to
be treated, the condition of the subject and other relevant medical
facts that may modify the activity of the drug or the response of
the subject, as is well-known by those skilled in the art.
EXAMPLE 1
Preparation of (24R/S-1,25-dihydroxy-3,5-cyclovitamin D.sub.2
(compounds 4 and 5):
Freshly recrystallized p-toluenesulfonly chloride (50 mg) was added
to a solution of 33 mg (24R/S)-25-hydroxyvitamin D.sub.2 (1) in dry
pyridine (300 .mu.l). The reaction mixture was allowed to stand for
30 hours at 4.degree. C., poured into ice/saturated NaHCO.sub.3
with stirring and extracted with benzene. The combined extracts
were washed with NaHCO.sub.3, water, aqueous CuSO.sub.4 solution,
water, then dried over MgSO.sub.4. Removal of solvent under reduced
pressure gave a crude tosylate (2), which can be used directly for
the next reaction.
A mixture of product (2) and NaHCO.sub.3 (150 mg) in 10 ml of
anhydrous methanol was heated at 55.degree. C. for 8.5 h with
stirring, concentrated to Ca. 2 ml and diluted with 80 ml of
benzene. The benzene solution was washed with water, dried over
MgSO.sub.4 and evaporated. The oily product (3) was sufficiently
pure for the following oxidation step.
To a stirred suspension of 4 mg of SeO.sub.2 in 5 ml of dry
CH.sub.2 Cl.sub.2 was added 13.2 .mu.l of t-BuOOH. After 0.5 h, 40
.mu.l of anhydrous pyridine was added and the mixture was stirred
at room temperature until homogeneous. Dichloromethane (3 ml) was
then added, the solution was cooled to 0.degree. C. and the
cyclovitamin product (3) was added in 4.5 ml of CH.sub.2 Cl.sub.2.
After 15 min, the reaction was allowed to warm slowly to room
temperature and continued until almost all starting material was
consumed (Ca. 30 min). The mixture was transferred to a separating
funnel and shaken with 30 ml of 10% NaOH. Ether (150 ml) was added
and the phases were separated. The etheral phase was washed with
10% NaOH, water, dried over MgSO.sub.4 and evaporated in vacuo. The
oily residue was purified by preparative TLC (developed with 6:4
n-hexane-ethyl acetate). The isolated product (12 mg) represents a
mixture containing 1.alpha.-hydroxyclovitamin 4 and a lesser amount
of the corresponding 1.beta.-hydroxy derivative 5, and is
characterized by the following physical data: mass spectrum, m/e
442 (M.sup.+, 13), 424 (8), 410 (9), 392 (26), 352 (15), 269 (27),
135 (88), 59 (100); NMR (CDCl.sub.3) .delta. 0.55 (3H, s,
18-H.sub.3), 0.63 (1H, m, 3-H), 1.00 (3H, d, J=6.5 Hz, 28-H.sub.3),
1.05 (3H, d, J=6.5 Hz, 21-H.sub.3), 1.13 and 1.18 (6H, each s,
26-H.sub.3 and 27-H.sub.3), 3.26 (3H, s, 6-OCH.sub.3), 4.19 (1H, d,
J=9.5 Hz, 6 -H), .about.4.2 (1H, m, 1-H), 4.96 (1H, d, J=9.5 Hz,
7-H), 5.17 and 5.24 (2H, each m, 19-H.sub.2), 5.35 (2H, broad m,
22-H and 23-H).
EXAMPLE 2
(a) Cycloreversion of 1-hydroxycyclovitamins 4 and 5
A solution containing a mixture of 1-hydroxycyclovitamins 4 and 5
(6 mg) in glacial acetic acid (0.5 ml) was heated at 55.degree. C.
for 15 min, cooled and poured carefully over ice-saturated
NaHCO.sub.3. The mixture was extracted with benzene and ether, and
the combined extracts were washed with saturated NaHCO.sub.3 and
water. The residue was chromatographed on a HPLC column (6.2
mm.times.25 cm Zorbax-Sil) using 3% 2-propanol in hexane as eluent.
Chromatography yielded a fraction (1.4 mg) containing
1.alpha.,25-dihydroxyvitamin D.sub.2 3-acetate (6a) and both
24-epimers of the corresponding 1.beta.,25-dihydroxy derivative 7
(peak collected at 90 ml) as well as a fraction (2.5 mg) containing
of (24R)-1.alpha.,25-dihydroxy-vitamin D.sub.2 3-acetate (6b) and
both 24-epimers of the 5,6-trans-1.alpha.,25-dihydroxyvitamin 8
(peak collected at 97 ml; 1:1 ratio at 6b and 8 was established by
NMR).
(b) Hydrolysis of 3.beta.-acetoxy group
A solution of the mixture containing 6a and 7 (1.1 mg) as obtained
above in 10% methanolic NaOH (1 ml) was heated at 55.degree. C. for
1 hour, then poured into the water and extracted with benzene,
ether and methylene chloride. The organic extracts were washed with
water, dried, combined and evaporated. HPLC of the residue (10%
2-propanol/hexane, 6.2.times.25 cm Zorbax-Sil column) afforded a
mixture of 24-epimers of 1.beta.,25-dihydroxyvitamin D.sub.2 11
(0.15 mg, peak collected at 52 ml) and a pure
1.alpha.,25-dihydroxyvitamin D.sub.2 10a (0.6 mg, peak collected at
59 ml). Rechromatography of 11 was performed on HPLC (4.6 mm
.times.25 cm Zorbax-Sil column using 4% 2-propanol in hexane as an
eluent). Pure compounds 11a and 11b were collected at 90 and 97
ml.
By analogous treatment of the mixture containing 6b and 8 (2.4 mg)
as obtained above with NaOH a mixture of
(24R)-1.alpha.,25-dihydroxyvitamin D.sub.2 (10b) and 24-epimers of
5,6-trans-1.alpha.,25-dihydroxyvitamin 12 (1.7 mg, 1:1 ratio of 10b
and 12) was obtained. Separation of the isomers was achieved on
HPLC (4.6.times.25 cm Zorbax-Sil column, 6% 2-propanol/hexane). The
chromatographic peaks for
(24S)-5,6-trans-1.alpha.,25-dihydroxyvitamin D.sub.2 (12a),
(24R)-1.alpha.,25-dihydroxyvitamin D.sub.2 (10b) and
(24R)-5,6-trans-1.alpha.,25-dihydroxyvitamin D.sub.2 (12b) were
partially overlapped (57 ml, 59 ml and 62 ml respectively) but
recycling afforded pure compounds.
(c) Cis to Trans Isomerization
The 5,6-cis form of the 1,25-dihydroxyvitamin D compounds thus
obtained can be converted to the trans compounds by treatment with
iodine. Thus, treatment of compound 10a in ether with a catalytic
amount of iodine (2% of the amount of 10a), while keeping the
solution under diffuse daylight for 1 hr, results in cis to trans
isomerization, and after HPLC purification (Zorbax-Sil column,
4.6.times.25 cm), 6% 2-propanol/hexane), the 5,6-trans-isomer 12a
was obtained. Under like conditions, compound 10b is isomerized to
12b. Similarly, compound 11a, upon treatment with iodine under the
above conditions provided a mixture of the 5,6-cis and
5,6-trans-isomer (11a, 13a) which when separated by HPLC
(Zorbax-Sil, 9.6.times.25 cm, 10% 2-propanol/hexane) gave 13a in
pure form, and treatment of compound 11b, under the same
conditions, gave the 5,6-trans compound 13b. These cis to trans
conversions are useful for correlating the respective C-24-epimers
of the major 5,6-cis products with the minor 5,6-trans compounds
resulting from the above solvolysis. Also, if the quantity of
starting material used in the above solvolysis reaction is low, so
as to make purification of the minor trans isomers from the
solvolysis mixture inefficient or unduly time consuming, the cis to
trans conversion may be used for preparing quantities of pure trans
material.
The following spectral data characterize the products obtained:
1.alpha.,25-dihydroxyvitamin D.sub.2 (10a): UV (EtOH)
.lambda..sub.max 265.5 nm, .lambda..sub.min 227.5 nm; mass
spectrum, m/e 428 (M.sup.+, 6), 410 (4), 352 (4), 287 (6), 269
(10), 251 (10), 152 (42), 134 (100), 59 (99); NMR (CDCl.sub.3)
.delta. 0.56 (3H, s, 18-H.sub.3), 1.01 (3H, d, J=6.5 Hz,
28-H.sub.3), 1.04 (3H, d, J=6.5 Hz, 21-H.sub.3), 1.14 and 1.18 (6H,
each s, 26-H.sub.3 and 27-H.sub.3), 4.24 (1H, m, 3-H), 4.43 (1H, m,
1-H), 5.01 (1H, m, 19-H), 5.34 (3H, broad m, 19-H, 22-H and 23-H),
6.02 (1H, d, J=11 Hz, 7-H), 6.39 (1H, d, J=11 Hz, 6-H).
1.alpha.,25-dihydroxy-24-epivitamin D.sub.2 (10b): UV (EtOH)
.lambda..sub.max 265.5 nm, .lambda..sub.min 227.5 nm; mass
spectrum, m/e 428 (M.sup.+, 13), 410 (9), 352 (7), 287 (11), 269
(15), 251 (13), 152 (52), 134 (100), 59 (97).
1.beta.,25-dihydroxyvitamin D.sub.2 (11a): UV (EtOH)
.lambda..sub.max 263.5 nm, .lambda..sub.min 227 nm; mass spectrum,
m/e 428 (M.sup.+, 9), 410 (27), 392 (12), 352 (8), 334 (7), 269
(12), 251 (15), 152 (48), 135 (68), 134 (53), 59 (100).
1.beta.,25-dihydroxy-24-epivitamin D.sub.2 (11b): UV (EtOH)
.lambda..sub.max 263.5 nm, .lambda..sub.min 227 nm; mass spectrum,
m/e 428 (M.sup.+, 10), 410 (29), 392 (13), 352 (9), 334 (7), 269
(13), 251 (16), 152 (58), 135 (76), 134 (59), 59 (100).
5,6-trans-1.alpha.,25-dihydroxyvitamin D.sub.2 (12a): UV (EtOH)
.lambda..sub.max 273.5 nm, .lambda..sub.min 230 nm; mass spectrum,
m/e 428 (M.sup.+, 8), 410 (3), 287 (3), 269 (7), 251 (7), 152 (34),
134 (100), 59 (78).
5,6-trans-1.alpha.,25-dihydroxy-24-epivitamin D.sub.2 (12b): UV
(EtOH) .lambda..sub.max 273.5 nm, .lambda..sub.min 230 nm; mass
spectrum, m/e 428 (M.sup.+, 10), 410 (4), 352 (4), 287 (5), 269
(9), 251 (8), 152 (37), 134 (100), 59 (82).
5,6-trans-1.beta.,25-dihydroxyvitamin D.sub.2 (13a): UV (EtOH)
.lambda..sub.max 270 nm, .lambda..sub.min 229.5 nm; mass spectrum,
m/e 428 (11), 410 (5), 351 (4), 287 (4), 269 (12), 251 (11), 152
(67), 135 (100), 134 (75), 59 (72).
5,6-trans-1.beta.,25-dihydroxy-24-epivitamin D.sub.2 (13b): UV
(EtOH) .lambda..sub.max 270 nm, .lambda..sub.min 229.5 nm; mass
spectrum, m/e 428 (M.sup.+, 11), 410 (4), 351 (4), 287 (4), 269
(13), 251 (12), 152 (64), 135 (100), 134 (70), 59 (63).
EXAMPLE 3
Conversion of 1.alpha.,25-dihydroxyvitamins D.sub.2 (10a,b) to
1.beta.,25-dihydroxyvitamin D.sub.2 analogs (11a,b):
A solution of 1.alpha.,25-dihydroxyvitamin D.sub.2 10a (40 .mu.g)
in anhydrous ether (1 ml) was treated with activated MnO.sub.2 (6
mg) for 5 h at room temperature. The mixture was filtered (Celite),
evaporated and the residue was chromatographed on HPLC column (6.2
mm .times.25 cm Zorbax-Sil) using 10% of iPrOH/hexane mixture as an
eluent. 1-oxo-previtamin 14a [UV:.lambda..sub.max (Et.sub.2 O)
233.5, 297 nm] was collected at 26 ml. Compound 14a was then
reduced with LiALH.sub.4 in anhydrous ether (-23.degree. C., 40
min). Excess of reagent was decomposed with water, anhydrous
MgSO.sub.4 was added and inorganic material was filtered off. After
removal of the solvent, the organic residue was dissolved in EtOH
and refluxed for 2.5 hours under argon atmosphere. Products were
separated by HPLC (9.4 mm.times.25 cm Zorbax-Sil column) using 15%
iPrOH/hexane mixture. Pure 1.beta.,25-dihydroxyvitamin D.sub.2 11 a
was collected at 55 ml and 1.alpha.,25-dihydroxyvitamin D.sub.2 10a
at 63 ml (3:1 ratio of 11a and 10a).
The analogous reaction sequence, performed with
1.alpha.,25-dihydroxy-24-epivitamin D.sub.2 (10b) resulted in
formation of 1.beta.,25-dihydroxy-24-epivitamin D.sub.2 (11b).
The following Example and its accompanying schematic, in each of
which like compounds are designated by like numbers, illustrate a
method for preparing the starting materials for the process of the
present invention.
EXAMPLE 4
The C-22 aldehyde (1) is obtained by degradation of ergosterol
acetate (in which the ring B diene system has been protected by
Diels-Alder addition of 4-phenyl-1,2,4-triazoline-3,5-dione)
according to the procedure of Barton et al. J. Chem. Soc. (c) 1968
(1971). The i-ether aldehyde (4) is obtained from stigmasterol by
the method of U.S. Pat. No. 2,623,052.
Synthesis of the Side Chain Fragment (Sulfone A)
To a stirred solution of 4-hydroxy-3-methylbutan-2-one (12.75 g;
0.125 mol) in pyridine (100 ml) is added p-toluenesulfonyl chloride
(p-TsCl) (33.25 g, 0.175 mol) in portions, and after standing for
14 hour at room temperature, the reaction mixture is poured into
water and extracted with CH.sub.2 Cl.sub.2. The extract is washed
several times with aqueous CuSO.sub.4 solution and water and then
dried over anhydrous sodium sulfate. Removal of solvent under
reduced pressure gives the crude tosylate which is used directly
for the next reaction.
Thiphenol (14 g) dissolved in DMF (100 ml) is treated with t-BuOK
(14 g). To this reagent, the tosylate is added and after 12 hour at
room temperature, the reaction mixture is poured into water and
extracted with CH.sub.2 Cl.sub.2. The extract is washed with
aqueous Na.sub.2 CO.sub.3 solution and water, then dried.
Evaporation of solvent gives an oily residue which is purified by
silica gel column chromatography. Pure phenyl sulfide is eluted
with benzene (yield 15 g).
To this phenyl sulfide derivative (15 g), in benzene (100 ml),
ethylene glycol (6 g) and p-TsOH (20 mg) is added and the reaction
mixture is heated under a Dean-Stark trap for 3 hour. After
cooling, it is extracted with Na.sub.2 CO.sub.3 solution and water,
then dried and the solvent is evaporated. The product, the desired
ketal, is chromatographically homogenous and can be used in the
next step without further purification.
Crude ketal in dichloromethane (250 ml) solution is treated with
m-chloroperbenzoic acid (m-CPBA) (80-85%, 27 g, added in portions)
while maintaining the temperature of the reaction mixture below
30.degree. C. After the addition of reagent, the reaction is
allowed to stand at room temperature with occasional shaking. When
the reaction reaches completion (about 1.5 hour), the aromatic
acids are removed by extraction with aqueous NH.sub.3, and the
organic layer is washed with water and dried. Evaporation of
solvent gives the oily sulfone (sulfone A) in essentially
quantitative yield (19 g). The product is substantially pure
(homogenous by TLC) and can be used without any further
purification; .sup.1 H-NMR; .delta.; 1.18 (d, J=7 Hz, 3H, CH.sub.3
--CH--), 1.19 (s, 3H, CH.sub.3 --C--), 3.84 (m, 4H, ketal-H),
7.3-7.6 and 7.6-7.9 (m, 3H+2H, aromatic protons); IR,
.gamma..sub.max.sup.KBr : 1305, 1147, 1082 cm.sup.-1 ; mass
spectrum, m/z (rel. intensity): 255 (M.sup.+ -Me, 21), 184 (66), 87
(92), 43 (100).
Coupling of Sulfone A to Aldehyde (1): Hydroxysulfone (2) and
Olefin (3).
Grignard reagent is prepared from Mg (535 mg; 22.22 mmol) and ethyl
bromide in ether (10 ml), and the virorously stirred solution is
treated with sulfone A (6 g; 2.22 mmol) in benzene (6 ml). The
precipitate formed is ground with a spatula, stirring is continued,
and after 15 min the aldehyde (1) (2.0 g) is added in benzene (10
ml). The reaction mixture is stirred at room temperature for 24
hour, then poured into aqueous (NH.sub.4).sub.2 SO.sub.4 solution
and extracted with benzene. The organic layer, after washing with
water, drying and evaporation gives an oily residue which is
chromatographed on silica gel. In the benzene-ether fractions
(8:2), excess sulfone is recovered (4.5 g); elution with
benzene-ether (3:1) affords unreacted aldehyde (1) (1.0 g); the
reaction products (2) are eluted with ethyl acetate.
The crude mixture of steroidal .alpha.-hydroxysulfones (2) is
dissolved in methanol (200 ml) saturated with Na.sub.2 HPO.sub.4.
Sodium amalgam (5.65%, 15 g) is added and the reaction mixture is
stirred at 4.degree. C. for 15 hour.
After completion of the Na/Hg reduction, mercury is removed by
filtration, and methanol by evaporation under reduced pressure,
water is added and the organic material is extracted with benzene.
After drying and evaporation of solvent, the oily residue is
chromatographed on a silica gel column. Elution with benzene-ether
(1:4) gives compound (3) a colorless foam; .sup.1 H-NMR, .delta.:
0.80 (s, 18-H), 0.97 (s, 19-H), 1.22 (s, 26-H), 3.93 (m, 4H,
ketal-H), 4.44 (m, 1H, 3-H), 5.25-5.45 (m, 2H, 22-H and 23-H), 6.23
and 6.39 (doublets, J=8 Hz, 2.times.1H, 7-H and 6-H), 7.25-7.45 (m,
5H, --C.sub.6 H.sub.5); IR, .gamma..sub.max.sup.CHCl13 : 3603
(O--H), 1749, 1692 (C.dbd.O), 1406, 1038 cm.sup.-1 ; mass spectrum,
m/z: 440 (M.sup.+ -triazoline, 24), 87 (100).
(To increase yield, unreacted aldehyde (1), as recovered above, can
be recycled through the sulfone addition, and the resulting
.alpha.-hydroxy sulfones (2) are then, as above, treated with
sodium amalgam in buffered methanol to provide additional olefin
(3). The above reactions are preferably conducted under an inert
atmosphere, such as argon.)
Coupling of Sulfone A to Aldehyde (4): Hydroxysulfone (5) and
Olefin (6).
Grignard reagent is prepared from Mg (75 mg, 3.1 mmol) and ethyl
bromide in ether (10 ml). To the stirred solution of ethyl
magnesium bromide, sulfone A (891 mg; 3.3 mmol) in benzene (5 ml)
is added. After stirring the resulting suspension at room
temperature for 15 min, a solution of aldehyde (4) (290 mg) in
benzene (5 ml) is added. The reaction is continued for 2.5 h, then
quenched with saturated (NH.sub.4).sub.2 SO.sub.4 solution (5 ml)
and diluted with ether. The separated organic layer is washed with
water, dried, and evaporated. The oily residue containing (5) is
treated with acetic anhydride (2 ml) and pyridine (2 ml). The
reaction mixture is allowed to stand for 24 hours, poured into
water and extracted with benzene. The benzene extract is washed
with an aqueous solution of CuSO.sub.4, water, dried, and
evaporated. The crude product [the acetate of (5)] is dissolved in
methanol saturated with Na.sub.2 HPO.sub. 4 and sodium amalgam
(5.65%, 8 g) is added. The reaction mixture is stirred at 4.degree.
C. for 16 hours. After the reaction, mercury is removed by
filtration, methanol is evaporated, and water and benzene are added
to dissolve the residue. The benzene layer is dried and evaporated.
The oily residue is chromatographed over silica gel. Elution with
benzene-ether mixture (93:7) affords compound (6) (206 mg; 54%),
.sup.1 H-NMR, .delta.: 0.74 (s, 18-H), 1.04 (s, 19-H), 1.25 (s,
26-H), 2.78 (m, 1H, 6--H), 3.34 (s, 3H, --OCH.sub.3), 3.97 (m, 4H,
ketal-H), 5.25-5.45 (m, 2H, 22-H and 23-H), IR,
.gamma..sub.max.sup.KBr : 3470 (O--H), 1095 cm.sup.-1 ; mass
spectrum, m/z (rel. intensity): 456 (M.sup.+, 1), 441 (M.sup.+ -Me,
45), 87 (100). It should be noted that the acetylation step
described above is not essential and may be omitted if desired;
i.e. the hydroxysulfone (5) may be submitted directly to
Na/Hg-reduction, as in Example 3. The above reactions are
preferably conducted under an inert atmosphere, e.g. argon.
Removal of PTAD-protecting Group: 5,7-Diene (7).
A mixture of the compound (3) (1 g) and lithium aluminum hydride
(1.8 g) in THF (120 ml) is heated under reflux for 10 hours. After
cooling, excess reagent is destroyed with a few drops of water, and
the mixture is dried over anhydrous MgSO.sub.4, filtered, and
solvent is evaporated to give colorless crystalline material. Crude
diene 7 is repeatedly crystallized from ethanol; first and second
crops combined give 415 mg of (7). The mother liquor is
chromatographed on silica gel column, to give with benzene-ether
(7:3), an additional 120 mg of (7); total yield 535 mg (79%); m.p.
132.degree.-134.degree. C. (from ethanol), .sup.1 H-NMR, .delta.:
0.63 (s, 18-H), 0.95 (s, 19-H), 1.23 (s, 26-H), 3.63 (m, 1H, 3-H),
3.95 (m, 4H, ketal-H), 5.20-5.50 (m, 3H, 22-H, 23-H and 7-H), 5.57
(m, 1H, 6-H); IR, .gamma..sub.max.sup.KBr : 3430 (O-H), 1063, 1038
cm.sup.-1 ; mass spectrum, m/z (rel. int.): 440 (M.sup.+, 50), 407
(M.sup.+ -H.sub.2 O--Me, 11), 87 (100); UV,
.lambda..sub.max.sup.EtOH : 282 nm (.epsilon.=11,000).
Irradiation of Compound (7): Previtamin Analog (8).
A solution of diene (7) (50 mg) in 150 ml of benzene-ether (1:4) is
cooled on ice and deoxygenated with argon for 20 min. The reaction
mixture is irradiated under argon atmosphere for 18 min with a
mercury arc lamp (Hanovia SA-1) fitted with a Vycor filter. The
solvent is evaporated and the residue is chromatographed on HPLC
(6.2 mm.times.25 cm microparticulate silica gel, 4 ml/min, 1400
psi) and eluted with 2% 2-propanol in hexane to yield 22 mg (44%)
of previtamin (8); .sup.1 H-NMR; .delta.: 0.73 (s, 18-H), 1.24 (s,
26-H), 1.64 (s, 19-H), 3.96 (m, 5H, ketal-H and 3-H), 5.35 (m, 2H,
22-H and 23-H), 5.50 (m, 1H, 9-H), 5.69 and 5.94 (doublets, J=11.5
Hz, 2.times.1H, 6-H and 7-H); UV, .lambda..sub.max.sup.EtOH : 263
nm (.epsilon.=8,900).
Isomerization of (8) to the Vitamin-Analog (9).
Previtamin 8 (22 mg) is dissolved in ethanol (40 ml) and heated
under reflux for 150 min (argon atmosphere). The product is
purified by HPLC to yield 18 mg (82%) of the pure vitamin (9);
.sup.1 H-NMR, .delta.: 0.75 (s, 18-H), 1.24 (s, 26-H), 3.94 (m, 5H,
ketal-H and 3-H), 4.81 and 5.04 (2 narrow m, 2.times.1H, 19(Z)- and
19(E)-H, 5.33 (m, 2H, 22-H and 23-H), 6.03 (d, J=11 Hz, 1H, 7-H),
6.22 (d, J=11 Hz, 1H, 6-H); mass spectrum, m/z (rel. int.): 440
(M.sup.+, 17), 87 (100), UV, .lambda..sub.max.sup.EtOH : 265 nm
(.epsilon.=17,000).
Hydrolysis of the ketal: Keto-Vitamin D.sub.2 -Analog (10).
To the solution of compound (9) (18 mg) in ethanol (35 ml),
p-toluenesulfonic acid (7.5 mg) in water (1 ml) is added and the
reaction mixture is heated under reflux for 90 min (the reaction
course is monitored by HPLC). The solvent is evaporated, the
residue is dissolved in benzene and extracted with water. The
benzene solution is dried (anhydrous MgSO.sub.4), and evaporated to
yield product (10) (16 mg; 99%). .sup.1 H-NMR, .delta.: 0.57 (s,
18-H), 1.04 (d, J=7 Hz, 21-H), 1.13 (d, J=7 Hz, 28-H), 2.12 (s, 3H,
26-H), 3.10 (m, 1H, 24-H), 3.96 (m, 1H, 3-H) , 4.82 and 5.05 (2
narrow m, 2.times.1H, 19(Z)- and 19(E)-H), 5.2-5.5 (m, 2H, 22-H and
23-H), 6.03 (d, J=11.5 Hz, 1H, 7-H), 6.22 (d, J=11.5 Hz, 1H, 6-H),
IR, .gamma..sub.max.sup.CHCl 3: 3596 (O--H), 1709 cm.sup.-1
(C.dbd.O); mass spectrum, m/z (rel. int.): 396 (M.sup.+, 41), 363
(M.sup.+ -H.sub.2 O-Me, 13), 271 (M.sup.+ -side chain, 16), 253
(m.sup.+ -side chain-H.sub.2 O, 23), 136 (100), 118 (95); UV,
.lambda..sub.max.sup.EtOH : 265 nm (.epsilon.=17,900).
Reaction of Ketone (10) with Methylmagnesium Iodide: 25-OH-D.sub.2,
(11a), and its Epimer (11b).
Grignard reagent is prepared from magnesium (240 mg) and methyl
iodide in anhydrous ether (20 ml). To one-tenth of this solution (2
ml; 0.5M solution of CH.sub.3 MgI) ketone (10) (16 mg; 0.04 mmol)
in ether (2 ml) is added. The reaction mixture is stirred at room
temperature for 2 hours under an inert atmosphere, then quenched
with aqueous solution of NH.sub.4 Cl, diluted with benzene and
washed with water. The organic layer is separated, dried and
evaporated. The crude product is first purified by silica gel
column chromatography (elution with 20% ether in benzene) and the
mixture of (11a) and (11b) (16 mg; 96%) thereby obtained is then
repeatedly chromatographed on HPLC column using 2% 2-propanol in
hexane as an eluent to separate the 24-stereoisomers,
24-epi-25-OH-D.sub.2 (11b) and 25-OH-D.sub.2 (11a). Chromatography
and rechromatography of each stereoisomer yields 4 mg of (11b)
(collected at 68 ml), 4 mg of (11a) (collected at 74 ml) and 7 mg
of the mixture of both epimers. Treatment of 2 mg of the epimer
mixture with excess acetic anhydride in pyridine solution at room
temperature overnight followed by standard work-up yields the
corresponding 3-O-acetates.
25-OH-D.sub.2 (11a): [.alpha.].sub.D.sup.25 +56.8.degree. (C=0.2 in
EtOH); .sup.1 H-NMR, .delta.: 0.57 (s, 18-H), 1.00 (d, J=7 Hz,
28-H), 1.04 (d, J=7 Hz, 21-H), 1.15 and 1.17 (2 singlets, 26-H and
27-H), 3.95 (m, 1H, 3-H), 4.82 and 5.05 (2 narrow m, 2.times.1H,
19(Z)- and 19(E)-H), 5.23-5.43 (m, 2H, 22-H and 23-H), 6.05 and
6.22 (2 doublets, J=11 Hz, 2.times.1H, 7-H and 6-H); IR,
.gamma..sub.max.sup.KBr : 3401 (O--H), 1645, 1631 (C.dbd.C), 971
cm.sup.-1 (trans C.dbd.C); mass spectrum, m/z (rel. int.): 412
(M.sup. +, 63), 394 (M.sup.+ -H.sub.2 O, 10), 379 (M.sup.+ -H.sub.2
O-Me, 23), 271 (M.sup.+ -side chain, 37), 253 (M.sup.+ -side
chain-H.sub.2 O, 43), 136 (100), 118 (86), 59 (99), UV,
.lambda..sub.max.sup.EtOH : 265 nm (.epsilon.=17,950).
24-epi-25-OH-D.sub.2 (11b): [.alpha.].sub.D.sup.25 +50.7.degree.
(C=0.2 in EtOH), .sup.1 H-NMR, .delta.: 0.57 (s, 18-H), 0.99 (d,
J=Hz, 28-H), 1.03 (d, J=7 Hz, 21-H), 1.14 and 1.16 (2 singlets,
26-H and 27-H), 3.94 (m, 1H, 3-H), 4.82 and 5.03 (2 narrow m,
2.times.1H, 19(Z) and 19(E)-H), 5.20-5.40 (m, 2H, 22-H and 23-H),
6.04 and 6.22 (2 doublets, J=11 Hz, 2.times.1H, 7-H and 6-H), IR,
.gamma..sub.max.sup.KBr : 3401 (OH), 1643, 1630 (C.dbd.C), 971
cm.sup.-1 (trans C.dbd.C); mass spectrum, m/z (rel. int.): 412
(M.sup.+, 62) 394 (M.sup.+ -H.sub.2 O; 12), 379 (M.sup.+ -H.sub.2
O-Me, 31), 271 (M.sup.+ -side chain, 44), 253 (M.sup.+ -side
chain-H.sub.2 O, 55), 136 (100), 118 (67), 59 (38); UV,
.gamma..sub.max.sup.EtOH : 265 nm (.epsilon.=17,300).
It should be noted that from pure provitamin (7) further synthesis
(i.e. the irradiation, isomerization, deketalization and Grignard
reaction steps) may be accomplished without chromatographic
purification of any intermediate. Careful column chromatography on
silica gel before the final separation on HPLC removes all
by-products.
* * * * *